Cephalosporin in crystalline form

ABSTRACT

The present invention relates to cephalosporin of formula (I) in crystalline form. The compound of formula (I) in crystalline form is useful as antibiotics having potent and broad antibacterial activity; especially against methicillin resistant  Staphylococci  (MRSA) and  Pseudomonas aeruginosa .

This application is a Divisional of application Ser. No. 10/547,648filed Sep. 1, 2005 now U.S. Pat. No. 7,531,650.

The present invention relates to cephalosporin in crystalline form and aprocess for its preparation. Further, the present invention relates tothe use of said cephalosporin in crystalline form alone or incombination with other compounds or formulations of said cephalosporinin crystalline form as antibiotic compounds.

The cephalosporin of formula I

-   -   as well as the process for its preparation of the amorphous form        is know from EP 1087980 and EP 0849269.

The cephalosporin of the above formula and its sodium salt(cephalosporin of formula III) have the disadvantage of low stabilitydue to their amorphous form. The problem to be solved by the presentinvention was to provide a cephalosporin in a more stable form.

An object of the present invention is to provide cephalosporin offormula I in crystalline form which have a higher stability.

It has been surprisingly found that a cephalosporin salt in the form ofstable crystals can be obtained by crystallizing a cephalosporin in thepresence of an acid.

The present invention relates to cephalosporin of formula I incrystalline form

Further, the present invention relates to cephalosporin of formula I,which is a hydrochloride hydrate.

The present invention also relates to cephalosporin of formula I, whichis a hydrobromide or hydrobromide hydrate.

Further, the present invention relates to cephalosporin of formula II

The present invention also relates to cephalosporin in crystalline formof formula I and II.

Further, the present invention relates to a cephalosporin in crystallineform having peaks at the diffraction angles at degrees 2θ (CuK_(α)radiation) shown in table 1 (see below) in its powder X-ray diffractionpattern:

diffraction angle 2θ (°) Relative Intensity 6.9 (m) 7.3 (s) 9.3 (m) 9.8(w) 11.5 (m) 13.1 (m) 13.8 (w) 14.5 (vs) 14.9 (m) 15.4 (m) 15.7 (m) 16.6(m) 17.2 (m) 18.2 (m) 18.5 (m) 18.7 (m) 19.2 (w) 19.6 (m) 20.3 (m) 20.9(s) 21.4 (m) 21.8 (m) 22.2 (s) 22.7 (s) 23.0 (m) 24.8 (m) 27.1 (m) 28.6(m) (vs) = very strong; (s) = strong; (m) = medium; (w) = weak; (vw) =very weak

It has to be understood that due to small changes in the experimentaldetails, small deviations in the 2θ-values of the characteristic peaksin the X-ray powder diffraction patterns may occur.

The present invention also relates to a process for the preparation ofcephalosporin which process comprises

-   a) mixing an acid and an organic solvent, and adding the solution to    cephalosporin of formula III, and stirring the mixture; or-   b) mixing an acid and an organic solvent, and adding cephalosporin    of formula III to the solution, and stirring the mixture; or-   c) suspending cephalosporin of formula III in water and an acid and    stirring the mixture.

Further, the present invention relates to a cephalosporin obtainable bythe process mentioned above.

The present invention also relates to compositions comprisingcephalosporin as mentioned above.

Further, the present invention relates to cephalosporin compounds asmentioned above as medicament.

The present invention also relates to the use of cephalosporin compoundsas mentioned above for the preparation of a medicament for use asantiinfectiva.

Further, the present invention relates to formulations of abovementioned cephalosporin with:

-   -   1) basic salts (e.g. carbonate, hydrogen carbonate). The use of        co-solvents such as PEG, PPG, ethanol, propylene glycol, benzyl        alcohol or mixtures thereof.    -   2) The use of buffers and in-situ salt formers (e.g. citrate,        acetate, phosphate, carbonate, lysine, arginine, tromethamine,        meglumine, ethylenediamine, triethanolamine) alone or in        combination or with co-solvents or basic salts as described in        1).    -   3) The use of complexing agents (e.g. PVP, cyclodextrines,        dextrose) alone or in combination with principles as described        in 1) and 2).    -   4) The use of surfactants (e.g. polysorbate, pluronic, lecithin)        alone or in combinations with principles as described in 1), 2)        and 3).    -   5) The principles described in 1), 2), 3) and 4) may apply in        direct combination or as separate principle such as an        reconstitution solution, used for reconstitution of the        cephalosporin salt/s.

The present invention also relates to compositions containing amorphousparts of cephalosporin of formula I and/or II according to any one ofclaims 1 to 5 or 7 to 8, and amorphous parts of cephalosporin of formulaIII, and crystalline parts of cephalosporin of formula II according toany one of claims 2 to 5 or 7 to 8, to sum up to 100%.

Further, the present invention also relates to the use of saidcephalosporin in crystalline form alone or in combination with othercompounds or formulations of said cephalosporin in crystalline form asantibiotic compounds.

The present invention also relates to a pharmaceutical preparationcontaining a compound as described above and a therapeutically inertcarrier, particularly for the treatment and prophylaxis of infectiousdiseases.

The term “crystallinity” or “crystalline” is used to describe the partof crystalline material compared to amorphous material and is estimatede.g. by the line shape and the background intensity in XRPD patterns aswell as from DSC measurements.

According to these methods, a crystallinity of 90% to 100% is estimated.In a more preferred embodiment the crystallinity is within the range of92% to 100%. In the most preferred embodiment the crystallinity iswithin the range of 95% to 100%.

The process for the preparation of compound of formula II may be carriedout in either an acid dissolved in organic solvents, an acid or inaqueous acid solutions. Preferred the process is carried out in aqueousacid solutions.

The term “acid”, as used within the present invention, means an acids,such as HBr or HCl, preferred HCl. The acid may be used in gaseous formor in dissolved (either in aqueous solution or in an organic solvent)form.

The term “organic solvents” as used within the present invention, meansorganic solvents such as C₁₋₄-alkanol (CH₃OH, C₂H₅OH, n-C₃H₇OH,i-C₃H₇OH, i-C₄H₉OH, n-C₄H₉OH, sec-C₄H₉OH), ketones (aceton,ethylmethylketone), ethers (THF, Dioxan) acetonitrile, preferably CH₃OH,C₂H₅OH, n-C₃H₇OH, i-C₃H₇OH, i-C₄H₉OH, n-C₄H₉OH, sec-C₄H₉OH, acetone oracetonitrile, most preferred MeOH.

The term “acid solution” as used within the present invention, means HBror HCl solutions, preferably aqueous HBr or HCl. The aqueous HClsolution in the concentration range of 1% to 30%, more preferred in theconcentration range of 5% to 25%, most preferred in the concentrationrange of 10% to 20%. The aqueous HBr solution in the concentration rangeof 1% to 62%, more preferred in the concentration range of 5% to 55%,most preferred in the concentration range of 8% to 20%.

The compound of formula I, II and III are useful as antibiotics havingpotent and broad antibacterial activity; especially against methicillinresistant Staphylococci (MRSA) and Pseudomonas aeruginosa.

Experimental Part:

Crystallization from Acid-Saturated Organic Solvents:

The sodium salt of cephalosporin of formula III was prepared accordingto the methods described in EP 1087980 and EP 0849269.

The crystallization experiments were carried out in that the acid(either in gaseous form or aqueous solution; preferred HBr or HCl; morepreferred HCl) was dissolved in organic solvents as defined above (mostpreferred methanol), and the solution was added to the cephalosporin offormula III and stirred up to 24 hours (preferably 3-20 hours, mostpreferred 4-7 hours). The resulting suspension is filtered, washed withan organic solvent (preferably acetone) and dried in an air flow for afew minutes.

TABLE 2 Compound No. III Solvent Yield Result 1 60.8 mg 5 ml MeOH, HClsaturated 32 mg Crystalline 1 ml water, 23° C. 2 60.8 mg 6 ml MeOH, HClsaturated, 25 mg Crystalline 23° C. 3  103 mg 15 ml MeOH, HCl saturated73 mg Crystalline (room temperature)

The reaction is carried out at a temperature in the range of 0-30° C.,preferred 5-25° C., most preferred 15-25° C.

The crystalline material obtained contained at least 50% of crystallinematerial.

Crystallization experiments in an acid (preferred HBr or HCl; morepreferred HCl), dissolved in organic solvents as defined above (mostpreferred methanol), led, according to DSC, elemental microanalytics,X-ray powder diffraction and Raman spectroscopy, to a crystallinecephalosporin of formula II.

The following examples and FIG. 1 are provided to aid the understandingof the present invention.

FIG. 1 shows Powder X-ray Diffraction Pattern of crystalline form ofcephalosporin of formula II (CuK_(α) radiation)

CRYSTALLIZATION FROM ACID SOLUTION

The following table shows a series of crystallization experiments insuspension.

The crystallization experiments were carried out in that cephalosporinof formula III is suspended in water and an acid (in gaseous form or inaqueous solution; preferred HBr or HCl; more preferred HCl). Theresulting suspension is stirred up to 24 hours (preferably 3-20 hours,most preferred 4-7 hours), filtered, washed with an organic solvent(preferably acetone) and dried in an air flow for a few minutes.

TABLE 3 Compound No. III Solvent Yield* Result 4 100 mg 1.6 ml water +0.4 ml HBr (48% in 40 mg Crystalline water) 5 61 mg 0.3 ml water + 6 mlHCl (25%) 27 mg Crystalline additionally 4 × 1 ml water, 23° C. 6 112 mg0.5 ml water + 10 ml HCl (25%), 56 mg Crystalline 15° C. 7 69 mg 0.3 mlwater + 4 ml HCl (25%), 26 mg Crystalline 20° C. + 4 ml HCl (32%), 20°C. 8 81 mg 1.4 ml water + HCl (25%), 23° C. 49 mg Crystalline 9 201 mg20 ml HCl (7.4%/2 N), 23° C. 181 mg Crystalline 10 151 mg 30 ml HCl(12.5%), 23° C. 136 mg Crystalline 11 150 mg 30 ml HCl (12.5%), 5° C.187 mg Crystalline 12 150 mg 15 ml HCl (12.5%), 20° C. 161 mgCrystalline 13 150 mg 30 ml HCl (7.4%/2 N), 23° C. 125 mg Crystalline 14100 mg 50 ml HCl (7.4%/2 N), 23° C. 70 mg Crystalline 15 101 mg 25 mlwater, 25 ml HCl (25%), 23° C. 81 mg Crystalline 16 102 mg 50 ml HCl(12.5%), 23° C. 82 mg Crystalline 17 202 mg 20 ml HCl (7.4%/2 N), 15° C.186 mg Crystalline *yield = mass after filtration, regardless of salt orhydrate formation, residual solvent (water) can not be excluded

The reaction is carried out at a temperature in the range of 0-30° C.,preferred 5-25° C., most preferred 15-25° C.

Crystallization experiments in water and an acid (preferred HBr or HCl;more preferred HCl) led, according to DSC elemental microanalytics andX-ray powder diffraction, to a crystalline cephalosporin of formula II.

Methods of Characterizing the Cephalosporin Material:

Dynamic Vapor Sorption:

In general, the DVS measurement indicates the investigated crystallinesample exists as a trihydrate form.

Elemental Microanalytics

Elemental microanalytics to demonstrate the existence(6R,7R)-7-[(Z)-2-(5-Amino-[1,2,4]thiadiazol-3-yl)-2-hydroxyimino-acetylamino]-8-oxo-3-[(E)-(R)-1′-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxycarbonyl)-2-oxo-[1,3′]bipyrrolidinyl-3-ylidenemethyl]-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylicacid Hydrochloride Trihydrate

TABLE 4 Analytical results of the investigated sample no. 17 (compoundII) are summarized below: element C H N S Cl O atomic weight 12.01 1.0014.01 32.07 35.45 16.00 number of atoms 26 33 8 2 1 14 mr(atoms) 312.2633.00 112.08 64.14 35.45 224.00 nominal % 39.99 4.23 14.35 8.21 4.5428.68 found % 39.23 4.20 14.06 7.86 4.56 29.20 difference % −1.89 −0.61−2.04 −4.30 0.45 1.80 Total mass: 780.93 assuming the compositionC₂₆H₂₆N₈O₁₁S₂•HCl•3H₂O

Methods of Proving/Characterizing the Presence of Crystalline Parts inthe Prepared Cephalosporin Material:

Differential Scanning Calorimetry (DSC):

DSC measurements were used to identify amorphous parts in samples of theHCl-salt.

DSC Investigation and X-Ray Powder Diffraction of Selected Samples

Selected samples have been investigated by DSC with respect to amorphousparts being present. In principle, two different kind of samples werefound: on the one hand samples showing decomposition between about 100°C. and 140° C., on the other hand a set of samples is characterized byan endothermic peak at about 149° C. and simultaneous decomposition.

Samples with an endothermic heat flow and presumably very smallamorphous parts according to DSC were further investigated by X-raypowder diffraction. In general, these samples showed similar diffractionpatterns but differed in the grade of crystallinity.

Determination of Storage Stability of Crystalline Material of Formula I

A crystalline and an amorphous sample of(6R,7R)-7-[(Z)-2-(5-Amino-[1,2,4]thiadiazol-3-yl)-2-hydroxyimino-acetylamino]-8-oxo-3-[(E)-(R)-1′-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxycarbonyl)-2-oxo-[1,3′]bipyrrolidinyl-3-ylidenemethyl]-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylicacid were stored at different temperatures for 24 hours, 28 days and for3 months. Results of HPLC analysis are summarized in table 4 to table 6.

TABLE 5 Storage of amorphous compound I and crystalline compound II for24 hours Area-% (HPLC) Area-% (HPLC) amorphous crystalline TemperatureRel. Humidity compound I compound II −20° C. Not defined 99.04 95.77  5Ca. 58% 99.05 95.7 25 Ca. 58% 98.85 95.75 40 Ca. 75% 98.25 95.45 60 Ca.75% 96.67 95.57

TABLE 6 Storage of amorphous compound I and crystalline compound II for28 days Area-% (HPLC) Area-% (HPLC) amorphous crystalline TemperatureRel. Humidity compound I compound II −20° C. Not defined 98.6 95.3  5Ca. 58% 98.1 95.2 25 Ca. 58% 96.4 94.9

TABLE 7 Storage of amorphous compound I and crystalline compound II for3 months Area-% (HPLC) Area-% (HPLC) amorphous crystalline TemperatureRel. Humidity compound I compound II −20° C. Not defined 97.6 94  5 Ca.58% 96.9 93.9 25 Ca. 58% 91.4 93.4

Storage for 24 hours revealed a very good stability of the crystallinecompound II in the whole temperature range of investigation. Theamorphous compound I decomposed significantly at temperatures above 5°C.

During 28 days a slight decomposition of crystalline compound II wasobserved at 25° C. In comparison, the content of compound I in theamorphous compound I decreased at 5° C. and even stronger at 25° C.

After 3 months, the amorphous compound I showed a slight decompositioneven at 5° C. The content of amorphous compound I strongly decreased at25° C. In contrast, the crystalline compound II showed no decompositionat 5° C. as compared to −20° C., at 25° C. a slight decomposition wasobserved.

The products in accordance with the invention can be used asmedicaments, for example, in the form of pharmaceutical preparations forenteral (oral) administration. The products in accordance with theinvention can be administered, for example, perorally, such as in theform of tablets, coated tablets, dragees, hard and soft gelatinecapsules, solutions, emulsions or suspensions, or rectally, such as inthe form of suppositories.

Pharmaceutical compositions containing these compounds can be preparedusing conventional procedures familiar to those skilled in the art, suchas by combining the ingredients into a dosage form together withsuitable, non-toxic, inert, therapeutically compatible solid or liquidcarrier materials and, if desired, the usual pharmaceutical adjuvants.

It is contemplated that the compounds are ultimately embodied intocompositions of suitable oral or parenteral dosage forms. Thecompositions of this invention can contain, as optional ingredients, anyof the various adjuvants which are used ordinarily in the production ofpharmaceutical preparations. Thus, for example, in formulating thepresent compositions into the desired oral dosage forms, one may use, asoptional ingredients, fillers, such as coprecipitated aluminumhydroxide, calcium carbonate, dicalcium phosphate, mannitol or lactose;disintegrating agents, such as maize starch; and lubricating agents,such as talc, calcium stearate, and the like. It should be fullyunderstood, however, that the optional ingredients herein named aregiven by way of example only and that the invention is not restricted tothe use hereof. Other such adjuvants, which are well known in the art,can be employed in carrying out this invention.

Suitable as such carrier materials are not only inorganic, but alsoorganic carrier materials. Thus, for tablets, coated tablets, drageesand hard gelatine capsules there can be used, for example, lactose,maize starch or derivatives thereof, talc, stearic acid or its salts.Suitable carriers for soft gelatine capsules are, for example, vegetableoils, waxes, fats and semi-solid and liquid polyols (depending on thenature of the active substance; no carriers are, however, required inthe case of soft gelatine capsules). Suitable carrier materials for thepreparation of solutions and syrups are, for example, water, polyols,saccharose, invert sugar and glucose. Suitable carrier materials forsuppositiories are, for example, natural or hardened oils, waxes, fatsand semi-liquid or liquid polyols.

As pharmaceutical adjuvants there are contemplated the usualpreservatives, solubilizers, stabilizers, wetting agents, emulsifiers,sweeteners, colorants, flavorants, salts for varying the osmoticpressure, buffers, coating agents and antioxidants.

The products in accordance with the invention can be used asmedicaments, for example, in the form of pharmaceutical preparations forparenteral administration, and for this purpose are preferably made intopreparations as lyophilisates or dry powders for dilution with customaryagents, such as water or isotonic common salt or carbohydrate (e.g.glucose) solution.

Depending on the nature of the pharmacologically active compound thepharmaceutical preparations can contain the compound for the preventionand treatment of infectious diseases in mammals, human and non-human, adaily dosage of about 10 mg to about 4000 mg, especially about 50 mg toabout 3000 mg, is usual, with those of ordinary skill in the artappreciating that the dosage will depend also upon the age, conditionsof the mammals, and the kind of diseases being prevented or treated. Thedaily dosage can be administered in a single dose or can be divided overseveral doses. An average single dose of about 50 mg, 100 mg, 250 mg,500 mg, 1000 mg, and 2000 mg can be contemplated.

1. A process for preparing a cephalosporin compound in crystalline form,said cephalosporin compound selected from the group consisting of acompound of the formula I:

and pharmaceutically acceptable salts thereof, comprising: a) providinga mixture of an acid, and a solvent selected from the group consistingof water and an organic solvent and mixing with a cephalosporin offormula III:

b) stirring said mixture; and c) thereafter separating saidcephalosporin compound from said mixture, wherein said separatedcephalosporin compound is in crystalline form.
 2. The process of claim1, wherein the organic solvent is selected from the group consisting ofC₁₋₄ alkanol, ketones, ethers and acetonitrile.
 3. The process accordingto claim 1, wherein said acid is hydrobromic or hydrochloric acid. 4.The process according to claim 3, wherein the organic solvent isselected from the group consisting of C₁₋₄ alkanol, ketones, ethers andacetonitrile.
 5. The process according to claim 4, wherein the organicsolvent is selected from the group consisting of CH₃OH, C₂H₅OH,n-C₃H₇OH, i-C₃H₇OH, i-C₄H₉OH, n-C₄H₉OH, sec-C₄H₉OH, acetone andacetonitrile.
 6. The process according to claim 4, wherein the organicsolvent is methanol.
 7. The process of claim 1 wherein said acid is HCl.8. The process of claim 1 wherein said acid is HBr.
 9. The process ofclaim 1 wherein the mixture is formed by forming a solution of said acidwith the organic solvent and adding said solution to said cephalosporinof formula III.
 10. The process of claim 1 wherein the mixture is formedby suspending said cephalosporin of Formula III in water containing saidacid.